DNA methylation in mammalian development and disease.

The DNA methylation field has matured from a phase of discovery and genomic characterization to one seeking deeper functional understanding of how this modification contributes to development, ageing and disease. In particular, the past decade has seen many exciting mechanistic discoveries that have substantially expanded our appreciation for how this generic, evolutionarily ancient modification can be incorporated into robust epigenetic codes. Here, we summarize the current understanding of the distinct DNA methylation landscapes that emerge over the mammalian lifespan and discuss how they interact with other regulatory layers to support diverse genomic functions. We then review the rising interest in alternative patterns found during senescence and the somatic transition to cancer. Alongside advancements in single-cell and long-read sequencing technologies, the collective insights made across these fields offer new opportunities to connect the biochemical and genetic features of DNA methylation to cell physiology, developmental potential and phenotype.

Extraembryonic gut endoderm cells undergo programmed cell death during development.

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.

Lambda3: homology search for protein, nucleotide, and bisulfite-converted sequences.

MOTIVATION: Local alignments of query sequences in large databases represent a core part of metagenomic studies and facilitate homology search. Following the development of NCBI Blast, many applications aimed to provide faster and equally sensitive local alignment frameworks. Most applications focus on protein alignments, while only few also facilitate DNA-based searches. None of the established programs allow searching DNA sequences from bisulfite sequencing experiments commonly used for DNA methylation profiling, for which specific alignment strategies need to be implemented. RESULTS: Here, we introduce Lambda3, a new version of the local alignment application Lambda. Lambda3 is the first solution that enables the search of protein, nucleotide as well as bisulfite-converted nucleotide query sequences. Its protein mode achieves comparable performance to that of the highly optimized protein alignment application Diamond, while the nucleotide mode consistently outperforms established local nucleotide aligners. Combined, Lambda3 presents a universal local alignment framework that enables fast and sensitive homology searches for a wide range of use-cases. AVAILABILITY AND IMPLEMENTATION: Lambda3 is free and open-source software publicly available at https://github.com/seqan/lambda/.

AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Dynamic antagonism between key repressive pathways maintains the placental epigenome.

DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape's molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Hijacking of transcriptional condensates by endogenous retroviruses.

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate 'hijacking' of transcriptional condensates in various developmental and disease contexts.

Acute lymphoblastic leukemia displays a distinct highly methylated genome.

DNA methylation is tightly regulated during development and is stably maintained in healthy cells. In contrast, cancer cells are commonly characterized by a global loss of DNA methylation co-occurring with CpG island hypermethylation. In acute lymphoblastic leukemia (ALL), the commonest childhood cancer, perturbations of CpG methylation have been reported to be associated with genetic disease subtype and outcome, but data from large cohorts at a genome-wide scale are lacking. Here, we performed whole-genome bisulfite sequencing across ALL subtypes, leukemia cell lines and healthy hematopoietic cells, and show that unlike most cancers, ALL samples exhibit CpG island hypermethylation but minimal global loss of methylation. This was most pronounced in T cell ALL and accompanied by an exceptionally broad range of hypermethylation of CpG islands between patients, which is influenced by TET2 and DNMT3B. These findings demonstrate that ALL is characterized by an unusually highly methylated genome and provide further insights into the non-canonical regulation of methylation in cancer.

RLM: fast and simplified extraction of read-level methylation metrics from bisulfite sequencing data.

SUMMARY: Bisulfite sequencing data provide value beyond the straightforward methylation assessment by analyzing single-read patterns. Over the past years, various metrics have been established to explore this layer of information. However, limited compatibility with alignment tools, reference genomes or the measurements they provide present a bottleneck for most groups to routinely perform read-level analysis. To address this, we developed RLM, a fast and scalable tool for the computation of several frequently used read-level methylation statistics. RLM supports standard alignment tools, works independently of the reference genome and handles most sequencing experiment designs. RLM can process large input files with a billion reads in just a few hours on common workstations. AVAILABILITY AND IMPLEMENTATION: https://github.com/sarahet/RLM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.

Epigenetic regulator function through mouse gastrulation.

During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type-specific transcription factors and ubiquitous epigenetic machinery, which recognizes universally available histone residues or nucleotides in a context-dependent manner1,2. The molecular functions of these regulators are generally well understood, but assigning direct developmental roles to them is hampered by complex mutant phenotypes that often emerge after gastrulation3,4. Single-cell RNA sequencing and analytical approaches have explored this highly conserved, dynamic period across numerous model organisms5-8, including mouse9-18. Here we advance these strategies using a combined zygotic perturbation and single-cell RNA-sequencing platform in which many mutant mouse embryos can be assayed simultaneously, recovering robust  morphological and transcriptional information across a panel of ten essential regulators. Deeper analysis of central Polycomb repressive complex (PRC) 1 and 2 components indicates substantial cooperativity, but distinguishes a dominant role for PRC2 in restricting the germline. Moreover, PRC mutant phenotypes emerge after gross epigenetic and transcriptional changes within the initial conceptus prior to gastrulation. Our experimental framework may eventually lead to a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9.

BACKGROUND: Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT). RESULTS: VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring. CONCLUSIONS: VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .

Nanopype: a modular and scalable nanopore data processing pipeline.

SUMMARY: Long-read third-generation nanopore sequencing enables researchers to now address a range of questions that are difficult to tackle with short read approaches. The rapidly expanding user base and continuously increasing throughput have sparked the development of a growing number of specialized analysis tools. However, streamlined processing of nanopore datasets using reproducible and transparent workflows is still lacking. Here we present Nanopype, a nanopore data processing pipeline that integrates a diverse set of established bioinformatics software while maintaining consistent and standardized output formats. Seamless integration into compute cluster environments makes the framework suitable for high-throughput applications. As a result, Nanopype facilitates comparability of nanopore data analysis workflows and thereby should enhance the reproducibility of biological insights. AVAILABILITY AND IMPLEMENTATION: https://github.com/giesselmann/nanopype, https://nanopype.readthedocs.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.